Autophagy is a cellular homeostasis pathway that has been implicated in numerous diseases. One of these diseases, Niemann-Pick disease type C (NPC), is an autosomal recessive, neurodegenerative disorder. Mutations in the NPC1 gene occur in 95% of patients, and the resultant NPC1 protein is misfolded and degraded or no longer capable of facilitating intracellular trafficking of lipids and cholesterol through the lysosome. There is currently no FDA-approved therapy for NPC, and thus there is a critical need to develop effective therapeutics to meet the needs of NPC patients. The long-term goal of this research is to address this need through the development of small-molecule autophagy modulators that restore lipid homeostasis in vivo. The overall objective of this proposal is to identify and optimize small molecules that modulate autophagy, improve the NPC phenotype in vitro, and restore lipid homeostasis in vivo while also extending life span. The rationale for this research is that various mechanisms of autophagy modulation, including early-stage inhibition, late-stage inhibition, and activation, have been reported to have potential therapeutic benefit in models of NPC. The central hypothesis of this research is that small molecules that modulate autophagy will alleviate cholesterol accumulation and extend life span of NPC mice. However, it is still unclear what mechanism of autophagy modulation is most beneficial, and this question will be a central focus of this research through unbiased identification of autophagy modulators that improve the NPC phenotype. This approach is innovative because it departs from the status quo of developing autophagy modulators and then exploring their effects in NPC and instead uses phenotypic screens to identify modulators that have a positive impact on NPC phenotypes with subsequent determination of the mechanism of autophagy modulation. Mass spectrometry imaging will be used as a novel method to determine modulator mechanism and to evaluate efficacy of autophagy modulators in vivo through the analysis of protein and lipid changes, which will also aid in the identification of biomarkers. The proposed research is significant because it will identify which mechanism of autophagy modulation is most beneficial in NPC, it will provide novel, small-molecule autophagy modulators with efficacy in NPC, and it will provide new strategies for the assessment of small-molecule mechanism in vivo without labeled probes. These advances will greatly contribute to the long-term goal of bringing new therapeutic options to NPC patients.